Method of treating melanocortin-4 receptor pathway-associated disorders

ABSTRACT

The disclosure is related to a method of treating a disorder, such as obesity or a condition associated with obesity, such as hyperphagia, in a subject using a melanocortin-4 receptor (MC4R) agonist.

BACKGROUND OF THE INVENTION

Melanocortin 4 receptor (MC4R) is a heterotrimeric G-protein-coupledreceptor, which transduces signals by activating second messengersystems, including adenylate cyclase. Expressed in hypothalamic nucleiand other neuronal and non-neuronal tissues, controlling feedingbehavior and energy homeostasis, MC4R integrates an agonist(anorexigenic) signal provided by the α-melanocyte stimulating hormone(α-MSH), and an antagonist (orexigenic) signal provided by theagouti-related peptide (AGPR). Signals from MC4R modulate feedingbehavior through secondary effector neurons.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery of novel MC4R agonists.They are useful, e.g., in treating disorders associated with obesity.Obesity disorders include, e.g., polygenic and monogenic obesitydisorders, including morbid obesity. Treatment of obesity results inreduced body weight mainly due to a loss of fat mass often due topharmacotherapy associated with a reduction in appetite and an increaseor normalization of energy expenditure. Improvements in comorbiditiescan include reduced insulin resistance and improvement in cardiovascularparameters, as a result of reduced blood pressure and/or heart rate.

Accordingly, in one aspect, the invention features a compound of FormulaI or II (or a pharmaceutically acceptable salt thereof). Formula I hasthe structure set forth below.

General Structure:

-   -   Aaa, Bbb=Selected from Cys, hCys, Pen; capable of establishing a        disulfide bridge or        -   Glu, Asp, Lys, Orn, Dpr, Dbu capable of establishing a            lactam bridge    -   Xxx=Asn, Gln, Ser, Thr    -   Yyy=Lys, Arg, D-Lys, D-Arg    -   A₁=H, Ac    -   A₂=OH, NH₂    -   Ac is acyl, e.g., acetyl.

In embodiments, the MC4R agonist is a compound of the followingstructure (Formula II) or a pharmaceutically acceptable salt thereof:

where Xxx is Asn, Gln, Ser, or Thr,where A₁ is H or Ac,where A₂ is OH or NH₂, andwhere Yyy is Lys, Arg, D-Lys, or D-Arg.

In embodiments, the MC4R agonist is chosen from one or more of thefollowing compounds, (or pharmaceutically acceptable salt thereof):

001554C (SEQ ID NO: 1) Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ 001555C(SEQ ID NO: 2) Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ 001556C(SEQ ID NO: 3) Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ 001574C:(SEQ ID NO: 4) Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ 001576C:(SEQ ID NO: 5) Ac-Arg-(Glu-Gln-D-Phe-Arg-Trp-Apr)-NH₂ (SEQ ID NO: 6)Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 7)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 8)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 9)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 10)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 11)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 12)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 13)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 14)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 15)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 16)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 17)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 18)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 19)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 20)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 21)Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 22)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 23)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 24)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 25)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 26)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 27)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 28)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 29)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 30)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 31)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 32)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 33)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 34)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 35)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 36)Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 37)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 38)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 39)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 40)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 41)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 42)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 43)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 44)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 45)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 46)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 47)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 48)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 49)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 50)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 51)Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 52)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 53)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 54)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 55)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 56)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 57)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 58)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 59)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 60)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 61)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 62)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 63)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 64)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ or (SEQ ID NO: 65)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂.

In another aspect, the invention provides, a method of eliciting anagonist effect from a melanocortin receptor in a subject in need thereofwhich comprises administering to said subject an effective amount of acompound of formula (I) or formula (II) as defined hereinabove, or apharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treating acondition or a disorder, e.g., a condition associated with obesity, byadministering a compound of formula (I) or formula (II) as definedhereinabove, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of treating ametabolic disease or medical condition accompanied by weight gain suchas obesity, feeding disorders and Prader-Willi Syndrome by administeringan effective amount of a compound of formula (I) or formula (II) asdefined hereinabove, or a pharmaceutically acceptable salt thereof.While not wishing to be bound by theory, it is believed that an agonistanti-obesity effect is elicited from the melanocortin receptor. In afurther aspect of the foregoing method, the disease or condition treatedis obesity. In yet a further aspect of the foregoing method, the diseaseor condition treated is a feeding disorder.

In another aspect, the present invention provides a method of decreasingfood intake, decreasing body weight or a combination thereof, byadministering an effective amount of a compound of formula (I) orformula (II) as defined hereinabove, or a pharmaceutically acceptablesalt thereof. In a preferred embodiment, the present invention providesa method of decreasing food intake, decreasing body weight or acombination thereof, by eliciting an agonist or antagonist effect from amelanocortin receptor by administering an effective amount of a compoundof formula (I) or (II) or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method of decreasingappetite without compromising body weight by administering an effectiveamount of a compound of formula (I) or formula (II) as definedhereinabove, or a pharmaceutically acceptable salt thereof. In anotheraspect, the present invention, provides a method of decreasing foodconsumption while decreasing body weight by administering an effectiveamount of a compound of formula (I) or formula (II) as definedhereinabove, or a pharmaceutically acceptable salt thereof. In anotheraspect, the present invention provides a method for increasing ornormalizing energy expenditure, e.g., facilitating and/or effectingweight loss due to a reduction in fat mass in the subject.

In embodiments, the condition or disorder is obesity, e.g., Prader-WilliSyndrome. In embodiments the agonist it administered to decrease foodintake in a subject in need thereof, or to decrease body weight.

In an embodiment a method described herein comprises ameliorating orimproving a clinical symptom or indicators associated with a disorderdescribed herein such as obesity, type-II diabetes, pre-diabeticcondition, blood level of haemoglobin A1C (Hb1Ac) above 6%,hyperinsulimenia, hyperlipidemia, insulin insensitivity, or glucoseintolerance; delaying, inhibiting or preventing the progression ofobesity and/or obesity related indications; or partially or totallydelaying, inhibiting or preventing the onset or development of obesityor a obesity related indication.

In embodiments, the method comprises administering the agonist in a unitdosage suitable for injection, e.g., subcutaneous injection, e.g., oraladministration, suppository mediated administration or administration ofthe compound by absorption through the skin, intranasal administrationor sublingual administration, to the subject.

In embodiments, the daily dosage is 0.1 mg to 10 mg. In embodiments, thedaily dosage is about 0.1 mg to about 7.5 mg. In embodiments, the dailydosage is about 0.1 mg to about 5 mg. In embodiments, the daily dosageis about 0.1 mg to about 2.5 mg. In embodiments, the daily dosage isabout 0.1 mg to about 2 mg. In embodiments, the daily dosage is about0.1 mg to about 1 mg. In embodiments, the daily dosage is about 0.2 mgto about 10 mg. In embodiments, the daily dosage is about 0.2 mg toabout 7.5 mg. In embodiments, the daily dosage is about 0.2 mg to about5 mg. In embodiments, the daily dosage is about 0.2 mg to about 2.5 mg.In embodiments, the daily dosage is about 0.2 mg to about 2 mg. Inembodiments, the daily dosage is about 0.2 mg to about 1.5 mg. Inembodiments, the daily dosage is about 0.2 mg to about 1 mg. Inembodiments, the daily dosage is about 0.3 mg to about 10 mg. Inembodiments, the daily dosage is about 0.3 mg to about 7.5 mg. Inembodiments, the daily dosage is about 0.3 mg to about 5 mg. Inembodiments, the daily dosage is about 0.3 mg to about 2.5 mg. Inembodiments, the daily dosage is about 0.3 mg to about 2 mg. Inembodiments, the daily dosage is about 0.3 mg to about 1.5 mg. Inembodiments, the daily dosage is about 0.3 mg to about 1 mg. Inembodiments, the daily dosage is about 0.25 mg (e.g., 0.25 mg) to about0.5 mg (e.g., 0.5 mg). In embodiments, the daily dosage is about 0.5 mg(e.g., 0.5 mg) to about 0.75 mg (e.g., 0.75 mg). In embodiments, thedaily dosage is about 0.25 mg (e.g., 0.25 mg). In embodiments, the dailydosage is about 0.5 mg (e.g., 0.5 mg). In embodiments, the daily dosageis about 0.75 mg (e.g., 0.75 mg) to about 1.25 mg (1.25 mg). Inembodiments, the daily dosage is about 1 mg (e.g., 1 mg). Inembodiments, the daily dosage is about 1.25 mg (e.g., 1.25 mg) to about2 mg (e.g., 2 mg). In embodiments, the daily dosage is about 1.5 mg(e.g., 1.5 mg). In embodiments, the daily dosage is about 2 mg (e.g., 2mg).

In embodiments, the method comprises administering the agonist in a unitdosage suitable for injection, e.g., subcutaneous injection, to thesubject.

In embodiments, the unit dosage comprises about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2mg of the agonist.

In embodiments, the unit dosage is disposed within a delivery device,e.g., a syringe (e.g., prefilled syringe), an implantable device, aneedleless hypodermic injection device, an infusion pump (e.g.,implantable infusion pump), or an osmotic delivery system.

In embodiments, the agonist is administered subcutaneously, e.g., bysubcutaneous injection.

In embodiments, the agonist is administered systemically e.g., by oraldosing or other route in a dose range, e.g., from 0.1 mg to 100 mg, from1 mg to 500 mg, or from 10 mg to 1000 mg, to attain effectivetherapeutic concentrations.

In embodiments, the agonist is administered systemically e.g. by oraldosing or other route in a dose range form in a dose range from 0.1 mgto 100 mg, from 1 mg to 500 mg, from 10 mg to 1000 mg, to attaineffective therapeutic concentrations.

In embodiments, the agonist is administered daily over a period of atleast 3 weeks, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 weeks or more, or at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months or more, or at least 1, 2, 3, 4years or more.

In embodiments, the subject is obese, e.g., severely obese.

In embodiments, the subject has early onset severe obesity.

In embodiments, the subject is hyperphagic.

In embodiments, the subject has a body mass index (BMI) greater than 25kg/m² (e.g., ≥25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50 kg/m² or greater) prior to administrationof the agonist, e.g., at the time the agonist is prescribed, or at thetime of the first administration.

In embodiments, the subject has a body mass index (BMI) greater than 35kg/m² (e.g., ≥36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50kg/m² or greater) prior to administration of the agonist, e.g., at thetime the agonist is prescribed, or at the time of the firstadministration.

In embodiments, the subject has a body mass index (BMI) greater than 40kg/m² (e.g., ≥41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55kg/m² or greater) prior to administration of the agonist, e.g., at thetime the agonist is prescribed, or at the time of the firstadministration.

In embodiments, the subject has a body mass index (BMI) greater than 45kg/m² (e.g., ≥46, 47, 48, 49, 50, 51, 52, 53, 54, 55 kg/m² or greater)prior to administration of the agonist, e.g., at the time the agonist isprescribed, or at the time of the first administration.

In embodiments, the subject has a BMI higher than the 85-95th percentileprior to administration of the agonist, e.g., at the time the agonist isprescribed, or at the time of the first administration.

In embodiments, the subject has failed one or more previous therapies,e.g., exercise, diet, surgery, including banded gastroplasty (such asvertical banded gastroplasty) or bariatric surgery or behavioraltherapies, prior to administration of the agonist, e.g., at the time theagonist is prescribed, or at the time of the first administration.

In embodiments, the subject has a lower body weight after administrationof the agonist than before administration of the agonist.

In embodiments, administration of the agonist results in a reduction ofweight in the subject compared to the weight of the subject beforetreatment of about 0.5 kg to 3 kg after 1 week of treatment, or about 1kg to 6 kg after 2 weeks of treatment, or about 2 kg to 12 kg after 4weeks of treatment, or about 4 kg to 24 kg after 8 weeks of treatment,or about 8 kg to 48 kg after 16 weeks of treatment.

In embodiments, administration of the agonist results in weight loss inthe subject at a rate of about 0.5-1 kg/week, 1-2 kg/week, e.g., about 2kg/week, e.g., over a period of 1-2 weeks of treatment or longer, 2-4weeks of treatment or longer, 4-8 weeks of treatment or longer, 8-16weeks of treatment or longer, 16-32 weeks or longer, or 32-64 weeks orlonger.

In embodiments, administration of the agonist results in a reduction inhunger level (e.g., a lower score on the Likert hunger scale, e.g., alower score by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 points) in thesubject compared to the hunger level of the subject before treatment,e.g., results in abolishment of hunger (e.g., a score of 0 on the Likerthunger scale) in the subject, e.g., after 1-2 weeks of treatment orlonger, 2-4 weeks of treatment or longer, 4-8 weeks of treatment orlonger, or 8-16 weeks of treatment or longer.

In embodiments, administration of the agonist results in nodetectable/significant decrease in resting energy expenditure (REE) inthe subject, e.g., over a period of 24 hours, one week, or 30 days orlonger, e.g., as compared to a control REE (e.g., the REE in the subjectprior to treatment or a predetermined REE, e.g., in subjects of similarpre-treatment BMI, e.g., when expressed as REE per kg of lean bodymass).

In embodiments, administration of the agonist results in an increase inresting energy expenditure (REE) in the subject, e.g., over a period of24 hours, one week, or 30 days, or longer e.g., as compared to a controlREE (e.g., the REE in the subject prior to treatment or compared to apredetermined REE, e.g., in subjects of similar pre-treatment BMI, whenexpressed as REE per kg of lean body mass, e.g., after a similar levelof weight loss has been attained by fasting).

In embodiments, administration of the agonist results in a reduction infood intake by the subject compared to a control (e.g., the food intakeof the subject prior to treatment), e.g., wherein the food intake isdaily food intake or food intake over a period of 24 hours, or one week.

In embodiments, administration of the agonist results in a reduction infood intake of at least 100 kilocalories, e.g., at least 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 1000 kilocalories or more, compared to a control(e.g., the food intake of the subject prior to treatment or apredetermined food intake level), e.g., wherein the food intake is dailyfood intake or food intake over a period of 24, hours, or one week.

In embodiments, administration of the agonist results in a reduction infood intake of at least 5 kcal/kg/day, e.g., 5, 10, 20, 30, 40, 50, 60,70, 80, or 90 or more kcal/kg/day. In embodiments, the reduction in foodintake is relative to the food intake at baseline. In embodiments, thebaseline food intake is at least 100 kcal/kg/day, e.g., for a pediatricsubject at about 1 year of age. In embodiments, the baseline food intakeis at least 40 kcal/kg/day, e.g., for a pediatric subject, e.g., in lateadolescence.

In embodiments, administration of the agonist results in a reduction inwaist circumference of the subject compared to a control (e.g., thewaist circumference of the subject prior to treatment), as measured 1,2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, administration of the agonist results in a reduction inwaist circumference of at least 2 cm (e.g., at least 2, 3, 4, 5, 6, 7,8, 9, 10 cm or more) in the subject compared to a control (e.g., thewaist circumference of the subject prior to treatment), as measured 1,2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, administration of the agonist results in no detectableincrease in blood pressure (e.g., diastolic and/or systolic bloodpressure) of the subject compared to the blood pressure of the subjectprior to treatment, as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks ormore after initiation of treatment.

In embodiments, administration of the agonist results in a reduction inblood pressure (e.g., diastolic and/or systolic blood pressure) of thesubject compared to the blood pressure of the subject prior totreatment, as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more afterinitiation of treatment.

In embodiments, administration of the agonist results in a reduction insystolic blood pressure of the subject of at least 1-3 mmHg (e.g., atleast 1, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 mmHg or more) compared tothe blood pressure of the subject prior to treatment, as measured 1, 2,3, 4, 5, 6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, administration of the agonist results in a reduction indiastolic blood pressure of the subject of at least 4 mmHg (e.g., atleast 4, 7, 7.5, 8, 8.5, 9, 9.5, 10 mmHg or more) compared to the bloodpressure of the subject prior to treatment, as measured 1, 2, 3, 4, 5,6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, the subject is a mammal, e.g., a human.

In an embodiment, the agonist is delivered by way of an implantabledevice, a needleless hypodermic injection device, an infusion pump(e.g., implantable infusion pump), or an osmotic delivery system.

In embodiments, the agonist is administered subcutaneously, e.g., bysubcutaneous injection.

In an aspect, provided herein is a unit dosage of an agonist describedherein, wherein the unit dosage contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5,3, 3.5, 4, 4.5, or 5 mg of the agonist.

In embodiments, the unit dosage contains 0.5 mg of agonist.

In embodiments, the unit dosage contains 1.0 mg of agonist.

In embodiments, the unit dosage contains 1.5 mg of agonist.

In embodiments, the unit dosage is suitable for injection, e.g.,subcutaneous injection.

In embodiments, the unit dosage is disposed in a delivery devicesuitable for injection, e.g., subcutaneous injection.

In embodiments, the unit dosage is disposed in a syringe or pen-typeinjector suitable for injection, e.g., subcutaneous injection.

In accordance with any method described herein, in embodiments, animproved symptoms comprise one or more of:

(a) a decrease in body weight;

(b) a decrease in waist circumference;

(c) a decrease in hunger level;

(d) a decrease in food intake level;

(e) a lack of decrease or an increase in resting energy expenditure;

(f) a decrease in insulin resistance;

(g) a decrease in sleep apnea; or

(h) a decrease in symptoms associated with the metabolic syndrome.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.Headings, sub-headings or numbered or lettered elements, e.g., (a), (b),(i) etc, are presented merely for ease of reading. The use of headingsor numbered or lettered elements in this document does not require thesteps or elements be performed in alphabetical order or that the stepsor elements are necessarily discrete from one another. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein “about” and “approximately” generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a givenvalue or range of values.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, or knowledge of (e.g., knowledge of the sequence or mutationalstate of) a genotype or a nucleic acid or polypeptide, by “directlyacquiring” or “indirectly acquiring” the physical entity, value, orknowledge. “Directly acquiring” means performing a physical process(e.g., performing a synthetic or analytical method) to obtain thephysical entity, value, or knowledge. “Indirectly acquiring” refers toreceiving the physical entity, value, or knowledge from another party orsource (e.g., a third party laboratory that directly acquired thephysical entity, value, or knowledge). Directly acquiring a physicalentity includes performing a process that includes a physical change ina physical substance, e.g., a starting material. Exemplary changesinclude making a physical entity from two or more starting materials,shearing or fragmenting a substance, separating or purifying asubstance, combining two or more separate entities into a mixture,performing a chemical reaction that includes breaking or forming acovalent or non-covalent bond. Directly acquiring a value or knowledgeincludes performing a process that includes a physical change in asample or another substance. Examples include performing an analyticalprocess which includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

As used herein, the term “obese” refers to a subject having a body massindex (BMI) within the ranges defined as “obese” by the Center forDisease Control (see, e.g., URL.cdc.gov/obesity/defining.html andwww.cdc.gov/obesity/childhood/defining.html, last accessed on Aug. 19,2015) or as defined by “Clinical Guidelines on the Identification,Evaluation, and Treatment of Overweight and Obesity in Adults” from theNational Institutes of Health. BMI is obtained by dividing a subject'sweight, e.g., in kilograms (kg) by the square of the subject's height,e.g., in meter (m). For example, an adult who has a BMI of 30 kg/m² orhigher is considered obese. For example, an adult with a BMI of 25.0 to29.9 kg/m² is considered overweight; an adult with a BMI of 18.5 to 24.9kg/m² is considered to have a normal or healthy weight range; and anadult with a BMI of less than 18.5 kg/m² is considered to beunderweight. For example, an adult having a height of 5 feet, 9 incheswith a body weight of 203 pounds or more is considered obese. Forchildren and teens, obese refers to a subject having a BMI at or abovethe 85^(th) to 95^(th) percentile for children and teens of the same ageand sex.

A “severely obese” subject or a subject having “severe obesity” refersto a subject having a BMI of 35 kg/m² or higher, e.g., 40 kg/m² orhigher. For example, a severely obese subject is over 100% over theideal (normal, healthy) body weight.

As used herein “early onset”, e.g., as in early onset obesity, refers toan onset (e.g., first occurrence of one or more symptoms of a disorder,e.g., a disorder described herein, e.g., obesity) that occurs in asubject before adulthood, e.g., during childhood, e.g., when the subjectis less 18 years of age or younger (e.g., 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year of age or younger, or duringadolescence, e.g., when the child is younger than 12 years of age orwhen the child is younger than 6 years of age).

As used herein, the term “metabolic syndrome” refers to a group ofsymptoms that occur together and increase the risk for coronary arterydisease, stroke, and type 2 diabetes. According to the American HeartAssociation and the National Heart, Lung, and Blood Institute, metabolicsyndrome also referred to as Syndrome X) is present if a subject hasthree or more of the following signs:

1) Blood pressure equal to or higher than 130/85 mmHg;

2) Fasting blood sugar (glucose) equal to or higher than 100 mg/dL;

3) Large waist circumference (length around the waist):

-   -   Men—40 inches or more;    -   Women—35 inches or more;

4) Low HDL cholesterol:

-   -   Men—under 40 mg/dL;    -   Women—under 50 mg/dL;

5) Triglycerides equal to or higher than 150 mg/dL.

Metabolic syndrome can be diagnosed by testing subject's blood pressure,blood glucose level, HDL cholesterol level, LDL cholesterol level, totalcholesterol level, and triglyceride level.

As used herein, the term “agonist” refers to any chemical compound,either naturally occurring or synthetic, that, upon interacting with(e.g., binding to) its target, e.g., MC4R, raises the signaling activityof MC4R above its basal level. An agonist can be a superagonist (i.e. acompound that is capable of producing a greater maximal response thanthe endogenous agonist for the target receptor, and thus has an efficacyof more than 100%), a full agonist (i.e. a compound that elicits amaximal response following receptor occupation and activation) or apartial agonist (i.e. a compounds that can activate receptors but areunable to elicit the maximal response of the receptor system).

As used herein “treating” includes achieving one or more of thefollowing results: reducing the body weight (as measured, for example,by a body mass index (BMI) and/or body weight), e.g., compared to acontrol (e.g., body weight before treatment or a predetermined bodyweight); reducing the waist circumference, e.g., compared to a control(e.g., waist circumference before treatment or a predetermined waistcircumference); reducing the hunger level, e.g., compared to a control(e.g., hunger level before treatment or a predetermined hunger level);increasing the resting energy expenditure (REE), e.g., compared to acontrol (e.g., REE before treatment or a predetermined REE); decreasingthe food intake, e.g., compared to a control level (e.g., beforetreatment or a predetermined food intake); ameliorating or improving aclinical symptom or indicators associated with a disorder describedherein such as obesity, type-II diabetes, pre-diabetic condition, bloodlevel of haemoglobin A1C (Hb1Ac) above 6%, hyperinsulimenia,hyperlipidemia, insulin insensitivity, or glucose intolerance; delaying,inhibiting or preventing the progression of obesity and/or obesityrelated indications; or partially or totally delaying, inhibiting orpreventing the onset or development of obesity or a obesity relatedindication. Delaying, inhibiting or preventing the progression of theobesity includes for example, delaying, inhibiting or preventing theprogression of a subject having normal weight to obesity. Inembodiments, a control is a value of a parameter measured beforetreatment by a MC4R agonist described herein or a predetermined value.The term “treating” further includes partially or totally reducing therisk for coronary artery disease, stroke, and type 2 diabetes associatedwith the metabolic syndrome as well as ameliorating or improving aclinical symptom or signs of metabolic syndrome associated withmetabolic syndrome, such as any one or more of the five indicatorslisted above. For example, the term “treating” includes delaying,inhibiting or preventing the progression of parameters associated withthe metabolic syndrome, including insulin resistance, glucose clearanceand parameters of cardiovascular disease including heart rate and bloodpressure.

As used herein “inhibition” or “inhibits” can include a reduction in acertain parameter, such as a parameter described herein. For example,inhibition of a parameter, e.g., activity, can be at least 5%, 10%, 20%,30%, 40%, or more is included by this term. Thus, inhibition need not be100%.

“Prophylactic treatment” refers to treatment before onset of obesity toprevent, inhibit or reduce its occurrence.

As used herein, the term “subject” refers to a mammal, e.g., a human.Subject can also refer to an animal in need of veterinary treatment,e.g., companion animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like).

As used herein, the term “mutation” can refer to an altered nucleic acidsequence of a gene or fragment thereof compared to a wild-type sequence.For example, a mutation can include a point mutation, frame-shiftmutation, missense mutation, inversion, deletion, insertion, truncation,chromosomal translocation. In embodiments, a mutation can result in thegene or fragment thereof coding for a non-functional protein, a proteinwith reduced activity (or a partially functional protein), or a proteinwith altered activity. For example, a “loss of function” mutation refersto a mutation that results in the gene or fragment thereof coding for anon-functional protein, which has substantially reduced activitycompared to its wild-type counterpart (e.g., a non-functional proteinhas less than 60%, 50%, 40%, 30% 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1% or less activity than its wild-type counterpart). For example,“partial loss of function” mutation refers to a mutation that results inthe gene or fragment thereof coding for a partially functional protein,which has reduced activity compared to its wild-type counterpart (e.g.,a partially functional protein has less than 50% and greater than 10% ofthe activity of its wild-type counterpart).

As used herein “unit dosage” refers to a physically discrete unit suitedas unitary doses for a subject to be treated. Each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier.

As used herein “dosage” refers to a quantity or amount of a therapeuticagent. In some embodiments, a dosage is the amount administered to thesubject in a single administration, e.g., in a single injection, asingle infusion, or single administration of one or more unit dosages.In embodiments, a dosage is the amount administered to the subject inmultiple administrations, e.g., multiple injections, multiple infusions,or multiple administrations of one or more unit dosages. In otherembodiments, a dosage can refer to the total amount administered to thesubject in a certain time period, e.g., per day. In such examples, thedosage is typically referred to as “daily dosage” or dosage in terms ofquantity per day.

As used herein “hunger” or “hunger level” refers to a subject'sappetite, desire to consume food, or perceived need for food. Inembodiments, the hunger or hunger level of a subject can be quantifiedby using a scale to obtain a hunger score. In embodiments, the scale forhunger assigns a higher score for a subject that more frequently (e.g.,often or always) feels unbearable hunger and a lower score for a subjectthat less frequently (e.g., sometimes or never) feels unbearable hunger.See, e.g., Sibilia. Psychologicol Topics 19 (2010), 2, 341-354. Forexample, a Likert scale for hunger can be used that assigns scores from0 to 10 points (0=no hunger; 10=severe hunger). In other examples, aLikert scale for hunger can be used that assigns scores from 1 to 4points, where a subject who never feels unbearable hunger is assigned ascore of 1, where a subject who sometimes feels unbearable hunger isassigned a score of 2, where a subject who often feels unbearable hungeris assigned a score of 3, and where a subject who always feelsunbearable hunger is assigned a score of 4. See i.d.

Disorders

In accordance with the methods and compositions described herein, insome embodiments, a MC4R agonist, e.g., MC4R agonist described herein isused to treat a disorder, such as a metabolic disorder, e.g., obesity,hyperphagia, or metabolic syndrome.

Prader Willi Syndrome (PWS)

Prader Willi Syndrome (PWS) is a rare genetic disease with a prevalenceranging from approximately one in 8,000 to one in 25,000 patients in theU.S. A hallmark of PWS is severe hyperphagia—an overriding physiologicaldrive to eat—leading to severe obesity and other complications. Obesityis one of the greatest health threats to PWS patients, and hyperphagiaimpairs the ability of PWS patients to live independently, requiringcostly and constant supervision to prevent overeating. Withoutsupervision, these patients are likely to die prematurely as a result ofchoking, stomach rupture, or from complications caused by morbidobesity. Currently, there are no approved treatments for the obesity andhyperphagia associated with PWS.

Symptoms of PWS include infantile hypotonia with failure to thrive,rapid weight gain and overeating during childhood, as well asintellectual disability, developmental delay, short stature,hypogonadism. Diagnostic criteria for PWS are described, e.g., in Holmet al. Pediatrics 91(1993):398-402.

Outcomes

In embodiments, methods described herein result in one or more outcomes,including a reduction of weight (e.g., body weight), a reduction inhunger level, no detectable decrease in energy expenditure (e.g.,resting energy expenditure), an increase in energy expenditure (e.g.,resting energy expenditure), a reduction in daily/weekly/monthly foodintake, a reduction in waist circumference, no detectable increase inblood pressure, or a reduction in blood pressure in a subject, e.g.,relative to a control.

In embodiments, the control is the measurement of the parameter in thesubject prior to administration of (treatment with) a MC4R agonist. Inembodiments, the control is a predetermined value, e.g., the value ofthe parameter in an average obese human population, e.g., of like ageand gender as the subject; or the value of the parameter measured in thesubject at a previous time point (e.g., at a previous visit, e.g., to aphysician, medical facility or laboratory).

In embodiments, the outcome (e.g., the reduction, increase, nodetectable decrease, or no detectable increase in a given parameter) ismeasured in the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or moreafter initiation of treatment with a MC4R agonist. In other embodiments,the outcome (e.g., the reduction, increase, no detectable decrease, orno detectable increase in a given parameter) is measured in the subjectover a period of time (e.g., over a period of 1-2 weeks, 2-4 weeks, 4-6weeks, 6-8 weeks, 8-12 weeks, or 12-16 weeks or longer) during a courseof treatment.

In embodiments, methods described herein result in a reduction of weight(e.g., body weight) in the subject compared to a control (e.g., weightof the subject before treatment or a predetermined value, e.g., averageweight of an obese human population of like age and gender as thesubject not subjected to therapeutic intervention, or the weight of thesubject at a previous measurement, e.g., at a previous visit). Inembodiments, the reduction is about 0.5 to 1 kg to 0.5 to 3 kg after 1week of treatment, about 1 kg to 6 kg after 2 weeks of treatment, about2 kg to 12 kg after 4 weeks of treatment, about 4 kg to 24 kg after 8weeks of treatment, or about 8 kg to 48 kg after 16 weeks of treatment.In embodiments, the reduction is at a rate of loss of about 0.5 to 2kg/week, e.g., about 2 kg/week, e.g., over a period of 1-2 weeks oftreatment or longer, 2-4 weeks of treatment or longer, 4-8 weeks oftreatment or longer, 8-16 weeks of treatment, or 16-32 weeks oftreatment, or longer.

Measurement of weight, e.g., body weight, can be performed usingstandard methods in the art.

In embodiments, methods described herein result in a reduction in hungerlevel in the subject compared to a control (e.g., hunger level of thesubject before treatment or a predetermined hunger level, e.g., averagehunger level of an obese human population of like age and gender as thesubject or the hunger level of the subject at a previous measurement,e.g., at a previous visit). In embodiments, the methods described hereinresult in abolishment of hunger in the subject.

In embodiments, hunger is measured by a scale, such as a Likert hungerscale, which ranges from 0 to 10 and is described herein. Inembodiments, methods described herein result in a reduction in hungerscore in the subject compared to a control (e.g., hunger level of thesubject before treatment or a predetermined hunger level, e.g., averagehunger level of an obese human population of like age and gender as thesubject or the hunger level of the subject at a previous measurement,e.g., at a previous visit). In embodiments, methods described hereinresult in a lower score on the Likert hunger scale, e.g., a lower scoreby at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 points, compared to thecontrol (e.g., hunger level of the subject before treatment or apredetermined hunger level, e.g., average hunger level of an obese humanpopulation of like age and gender as the subject or the hunger level ofthe subject at a previous measurement, e.g., at a previous visit). Inembodiments, methods described herein result in a score of 0 on theLikert hunger scale after treatment.

In embodiments, the reduction in hunger level is measured/observed after1 to 2 weeks of treatment or longer, 2-4 weeks of treatment or longer,4-8 weeks of treatment or longer, or 8-16 weeks of treatment or longer.

REE is a measure of the basal metabolic rate of the subject and can bedetermined using methods such as those described in Chen et al. J. Clin.Endocrinol. Metab. 100.4(2015):1639-45. In embodiments, the REE can bedetermined by placing the subject in a whole-room indirect calorimeter(also called a metabolic chamber) at a certain time after treatment(e.g., after 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, or more weeks). Inembodiments, the REE is measured in 30-minute measurements periods, andin some cases, REE values from several 30-minute periods are averaged togenerate an average REE. In embodiments, the REE can be determined aftera 10-12 hour fasting period, at thermoneutrality (e.g., around 25 degC.), where the subject is awake without psychological or physicalstress. In embodiments, REE is measured in units of energy per unit time(e.g., kcal/h or kcal/day). In embodiments, the REE is measured relativeto kg lean body mass in a subject (e.g., REE/kg lean mass), e.g., asdescribed in the Examples.

In embodiments, methods described herein result in no change or nodecrease in energy expenditure, e.g., resting energy expenditure (REE),in the subject over an hourly, daily (e.g., in 24 hours), weekly (e.g.,in 7 days), or monthly (e.g., in 30 days) period compared to a controlREE (e.g., the REE in the subject prior to treatment or a predeterminedREE, e.g., average REE of an obese human population of like age andgender and normalized for weight as the subject or the REE of thesubject at a previous measurement, e.g., previous visit), e.g., asmeasured after 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, or more weeks oftreatment.

In embodiments, methods described herein result in no detectable changeor no detectable decrease in energy expenditure, e.g., resting energyexpenditure (REE) per kg lean body mass, in the subject over an hourly,daily (e.g., in 24 hours), weekly (e.g., in 7 days), or monthly (e.g.,in 30 days) period compared to the control REE (e.g., the REE in thesubject prior to treatment or a predetermined REE, e.g., average REE ofan obese human population of like age and gender as the subject or theREE of the subject at a previous measurement, e.g., previous visit),e.g., as measured after 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, or more weeksof treatment.

In embodiments, methods described herein result in an increase in energyexpenditure, e.g., resting energy expenditure (REE), in the subject overa hourly, daily (e.g., in 24 hours), weekly (e.g., in 7 days), ormonthly (e.g., in 30 days) period compared to a control REE (e.g., theREE in the subject prior to treatment or a predetermined REE, e.g.,average REE of an obese human population of like age and gender andnormalized for weight as the subject or the REE of the subject at aprevious measurement, e.g., previous visit), e.g., as measured after 3,4, 5, 6, 7 days, or 1, 2, 3, 4, or more weeks of treatment.

In embodiments, the increase in REE in the subject is at least 20kcal/day (e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150 kcal/day or more), e.g., as measured after 3, 4, 5, 6, 7days, or 1, 2, 3, 4, or more weeks of treatment.

In embodiments, the increase in REE in the subject is at least 2% (e.g.,at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% ormore), e.g., as measured after 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, ormore weeks of treatment, compared to the REE in the subject prior totreatment.

In embodiments, the REE in the subject (e.g., adult subject) aftertreatment with a MC4R agonist (e.g., after 3, 4, 5, 6, 7 days, or 1, 2,3, 4, or more weeks of treatment) is at least 1800 kcal/day (e.g., atleast 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050,2100, 2150, 2200, 2250, 2300, 2400 kcal/day, or more), e.g., for anadult subject. In embodiments, the REE in the subject (e.g., pediatricsubject) after treatment with a MC4R agonist (e.g., after 3, 4, 5, 6, 7days, or 1, 2, 3, 4, or more weeks of treatment) is at least 200kcal/day (e.g., at least 200, 225, 250, 275, 300, 325, 350, 375, 400,450, 500 kcal/day or more), e.g., for pediatric patients.

In embodiments, methods described herein result in a reduction in foodintake by the subject compared to a control (e.g., the food intake ofthe subject prior to treatment or a predetermined food intake level,e.g., the food intake of an average human obese population or the foodintake of the subject at a previous measurement, e.g., at a previousvisit), e.g., where the food intake is measured as daily food intake orfood intake over a period of 24 hours, or one week. In embodiments, thereduction is at least 100 kilocalories, e.g., at least 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,525, 550, 575, 600, 1000 kilocalories or more, e.g., for daily foodintake or food intake over a period of 24 hours, or one week, or 30 daysor for longer time periods, e.g., for an adult subject. In embodiments,mean food intake can decrease from a baseline at or above about 100kcal/kg/day to about 90, 80, 70, 60, 50, 40, 30, 20 or 10 kcal/kg/day orlower after treatment with a MC4R agonist, e.g., in a pediatric subjectat about 1 year of age. In embodiments, mean food intake can decreasefrom a baseline at or above about 40 kcal/kg/day to about 35, 30, 20 or10 kcal/kg/day or lower after treatment with a MC4R agonist, e.g., in apediatric subject in late adolescence.

Food intake can be determined by standard methods, e.g., as described inRutishauser. Pub. Health Nutr. 8.7A(2005):1100-07.

In embodiments, methods described herein result in a reduction in waistcircumference of the subject compared to a control (e.g., the waistcircumference of the subject prior to treatment or the waistcircumference of the subject at a previous measurement, e.g., previousvisit), as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more afterinitiation of treatment.

In embodiments, the reduction in waist circumference is at least 2 cm(e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 cm or more) in the subject(e.g., adult subject) compared to a control (e.g., the waistcircumference of the subject prior to treatment or a predetermined waistcircumference, e.g., the waist circumference of an average obese humanpopulation of like age and gender or the waist circumference of thesubject at a previous measurement, e.g., previous visit), as measured 1,2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, the waist circumference is measured using standardmethods. In embodiments, the waist circumference is the largestcircumference around a subject's mid-section, e.g., around a subject'sabdomen. In other embodiments, the waist circumference is measuredaround the natural waist (e.g., in between the lowest rib and the top ofthe hip bone), the umbilicus, or at the narrowest point of themidsection.

In embodiments, methods described herein result in no detectableincrease in blood pressure (e.g., diastolic and/or systolic bloodpressure) of the subject compared to a control blood pressure (e.g., theblood pressure of the subject prior to treatment or a predeterminedblood pressure, e.g., the blood pressure of an average obese humanpopulation of like age and gender or the blood pressure of the subjectat a previous measurement, e.g., previous visit), as measured 1, 2, 3,4, 5, 6, 7, 8, 9, 10 weeks or more after initiation of treatment.

In embodiments, methods described herein result in a reduction in bloodpressure (e.g., diastolic and/or systolic blood pressure) of the subjecta control blood pressure (e.g., the blood pressure of the subject priorto treatment or a predetermined blood pressure, e.g., the blood pressureof an average obese human population of like age and gender or the bloodpressure of the subject at a previous measurement, e.g., previousvisit), as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more afterinitiation of treatment.

In embodiments, the reduction in blood pressure, e.g., systolic bloodpressure, is at least 1, 2 or 3 mmHg (e.g., at least 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7 mmHg or more) compared to the blood pressure of thesubject prior to treatment, as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10weeks or more after initiation of treatment.

In embodiments, the reduction in blood pressure, e.g., diastolic bloodpressure, is at least 4 mmHg (e.g., at least 4, 7, 7.5, 8, 8.5, 9, 9.5,10 mmHg or more) compared to the blood pressure of the subject prior totreatment, as measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more afterinitiation of treatment.

In embodiments, the methods described herein do not result in an adverseeffect on heart rate or blood pressure.

Subjects

In accordance with any method described herein, in certain embodiments,the subject is obese, e.g., prior to administration of an agonistdescribed herein, e.g., at the time the agonist is prescribed, or at thetime of the first administration of the agonist. In embodiments, thesubject is a severely obese, pediatric or adult patient e.g., prior toadministration of an agonist described herein, e.g., at the time theagonist is prescribed or at the time of the first administration of theagonist. In embodiments, the subject is hyperphagic, e.g., prior toadministration of an agonist described herein, e.g., at the time theagonist is prescribed, or at the time of the first administration of theagonist.

In embodiments, the subject (e.g., adult subject) has a body mass index(BMI) greater than 25 kg/m² or 30 kg/m² (e.g., ≥25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 kg/m² or greater) prior to administration of the agonist, e.g.,at the time the agonist is prescribed, or at the time of the firstadministration.

In embodiments, the subject (e.g., pediatric subject) has a body massindex (BMI) higher than 85-95 percentile prior to administration of theagonist, e.g., at the time the agonist is prescribed, or at the time ofthe first administration.

In embodiments, the subject has a body weight of at least about 5 kg,e.g., at least about 5 kg, 10 kg, 20 kg, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 200, 205, 210, 215, 220 kg or greater, e.g., prior toadministration of the agonist, e.g., at the time the agonist isprescribed, or at the time of the first administration. In embodiments,the subject has a body weight of a least 20 kg, at least 60 kg, or atleast 100 kg, e.g., prior to administration of the agonist, e.g., at thetime the agonist is prescribed, or at the time of the firstadministration.

In embodiments, the subject is an adult, e.g., 18 years of age or older,e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, or older.

In embodiments, the subject is a pediatric subject, e.g., less 18 yearsof age or younger (e.g., 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 year of age or younger.

In yet another aspect, the present invention provides a method ofeliciting an agonist effect from a melanocortin receptor in a subject inneed thereof. The method comprises administering to the subject aneffective amount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of elicitingan agonist effect from a melanocortin receptor in a subject in needthereof. The method comprises administering to the subject an effectiveamount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof, wherein thecompound is a selective melanocortin 4 receptor agonist.

In a further aspect, the present invention provides a method ofeliciting an agonist or an antagonist effect from a melanocortinreceptor in a subject in need thereof. The method comprisesadministering to the subject an effective amount of a compound offormula (I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof, wherein the compound is a selectivemelanocortin 4 receptor agonist.

In yet another aspect, the present invention provides a method ofeliciting an agonist effect from a melanocortin receptor in a subject inneed thereof. The method comprises administering to the subject aneffective amount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof, wherein thecompound is a selective melanocortin 4 receptor agonist.

In another aspect, the present invention provides a method of treatingan acute or chronic inflammatory disease or medical condition such asgeneral inflammation, inflammatory bowel disease, brain inflammation,sepsis and septic shock by eliciting an agonist effect from amelanocortin receptor by administering an effective amount of a compoundof formula (I) or formula (II) as defined hereinabove, orpharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of treating adisease or medical condition with an autoimmune component such asrheumatoid arthritis, gouty arthritis and multiple sclerosis byeliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) orformula (II) as defined hereinabove, or pharmaceutically acceptablesalts thereof.

In another aspect, the present invention provides a method of treating ametabolic disease or medical condition accompanied by weight gain suchas obesity, feeding disorders and Prader-Willi Syndrome by eliciting anagonist effect from a melanocortin receptor by administering aneffective amount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof. In a furtheraspect of the foregoing method, the disease or condition treated isobesity. In yet a further aspect of the foregoing method, the disease orcondition treated is a feeding disorder characterized by hyperphagiayand/or obesity.

In another aspect, the present invention provides a method of decreasingfood intake, decreasing body weight or a combination thereof, byeliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) orformula (II) as defined hereinabove, or pharmaceutically acceptablesalts thereof. In an embodiment, the present invention provides a methodof decreasing food intake, decreasing body weight or a combinationthereof, by eliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) orformula (II), or pharmaceutically acceptable salts thereof.

In another embodiment, the present invention provides a method ofdecreasing food intake, decreasing body weight or a combination thereof,by eliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) or (II),or pharmaceutically acceptable salts thereof.

In another embodiment, the present invention provides a method ofdecreasing food intake, decreasing body weight or a combination thereof,by eliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) or (II),or pharmaceutically acceptable salts thereof. In another embodiment, thepresent invention provides a method of decreasing food intake,decreasing body weight or a combination thereof, by eliciting an agonisteffect from a melanocortin receptor by administering an effective amountof a compound of formula (I) or formula II, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of decreasingappetite without compromising body weight by administering an effectiveamount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof. In anotheraspect, the present invention provides a method of decreasing foodconsumption while decreasing body weight by administering an effectiveamount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of treating areproductive or sexual medical condition such as endometriosis, uterinebleeding, sexual dysfunction, erectile dysfunction and decreased sexualresponse in females by eliciting an agonist effect from a melanocortinreceptor by administering an effective amount of a compound of formula(I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of treating adisease or medical condition resulting from treatment or insult to anorganism such as organ transplant rejection, ischemia and reperfusioninjury, wounding and spinal cord injury, and weight loss due to amedical procedure selected from the group consisting of chemotherapy,radiation therapy, temporary or permanent immobilization and dialysis byeliciting an agonist effect from a melanocortin receptor byadministering an effective amount of a compound of formula (I) orformula (II) as defined hereinabove, or pharmaceutically acceptablesalts thereof.

In another aspect, the present invention provides a method of treating acardiovascular disease or medical condition such as hemorrhagic shock,cardiogenic shock, hypovolemic shock, cardiovascular disorders andcardiac cachexia by eliciting an agonist effect from a melanocortinreceptor by administering an effective amount of a compound of formula(I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of treating apulmonary disease or medical condition such as acute respiratorydistress syndrome, pulmonary fibrosis, chronic obstructive pulmonarydisease and asthma by eliciting an agonist effect from a melanocortinreceptor by administering an effective amount of a compound of formula(I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of enhancingimmune tolerance or treating allergies by eliciting an agonist effectfrom a melanocortin receptor by administering an effective amount of acompound of formula (I) or formula (II) as defined hereinabove, orpharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of treatingdermatological disease or medical condition such as psoriasis,erythropoietic protoporphyria, skin pigmentation depletion, acne andkeloid formation by eliciting an agonist effect from a melanocortinreceptor by administering an effective amount of a compound of formula(I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of treating abehavioral or central nervous system or neuronal disease or medicalcondition such as anxiety, depression, memory dysfunction andneuropathic pain by eliciting an agonist effect from a melanocortinreceptor by administering an effective amount of a compound of formula(I) or formula (II) as defined hereinabove, or pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention provides a method of treating arenal disease or medical condition such as renal cachexia andnatriuresis by eliciting an agonist effect from a melanocortin receptorby administering an effective amount of a compound of formula (I) orformula (II) as defined hereinabove, or pharmaceutically acceptablesalts thereof.

In another aspect, the present invention provides a method of modulatinga normalizing or homeostatic activity such as ovarian weight, placentaldevelopment, prolactin secretion, FSH secretion, intrauterine fetalgrowth, parturition, spermatogenesis, thyroxin release, aldosteronesynthesis and release, gonadal development, body temperature, bloodpressure, heart rate, vascular tone, brain blood flow, blood glucoselevels, sebum secretion, pheromone secretion, motivation, learning andbehavior, pain perception, neuroprotection and nerve growth by elicitingan agonist effect from a melanocortin receptor by administering aneffective amount of a compound of formula (I) or formula (II) as definedhereinabove, or pharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of modulatinga normalizing or homeostatic activity such as bone metabolism, boneformation and bone development by eliciting an agonist effect from amelanocortin receptor by administering an effective amount of a compoundof formula (I) or formula (II) as defined hereinabove, orpharmaceutically acceptable salts thereof.

In another aspect, the present invention provides a method of inhibitingalcohol consumption, for reducing alcohol consumption, for treatingalcoholism, or for treating alcohol abuse by eliciting an agonist effectfrom a melanocortin receptor by administering an effective amount of acompound of formula (I) or formula (II) as defined hereinabove, orpharmaceutically acceptable salts thereof. In a further aspect of theforegoing method, the compound is a selective melanocortin 4 receptoragonist.

In a further aspect, the present invention provides the use of atherapeutically effective amount of a melanocortin 4 receptor agonistcompound according formula (I) or formula (II) as defined hereinabove,or pharmaceutically acceptable salts thereof, for the manufacture of amedicament useful to treat a disease and/or medical condition selectedfrom the group consisting of acute and chronic inflammatory diseasessuch as general inflammation, inflammatory bowel disease, braininflammation, sepsis and septic shock; diseases with an autoimmunecomponent such as rheumatoid arthritis, gouty arthritis and multiplesclerosis; metabolic diseases and medical disorders accompanied byweight gain such as obesity, feeding disorders and Prader-WilliSyndrome; metabolic diseases and medical disorders accompanied by weightloss such as anorexia, bulimia, AIDS wasting, cachexia, cancer cachexiaand wasting in frail elderly; diabetes, diabetalogical relatedconditions and complications of diabetes such as retinopathy; neoplasticproliferation such as skin cancer and prostate cancer; reproductive orsexual medical conditions such as endometriosis and uterine bleeding inwomen, sexual dysfunction, erectile dysfunction and decreased sexualresponse in females; diseases or conditions resulting from treatment orinsult to the organism such as organ transplant rejection, ischemia andreperfusion injury, spinal cord injury and wounding, as well as weightloss caused chemotherapy, radiation therapy, temporary or permanentimmobilization or dialysis; cardiovascular diseases or conditions suchas hemorrhagic shock, cardiogenic shock, hypovolemic shock,cardiovascular disorders and cardiac cachexia; pulmonary diseases orconditions such as acute respiratory distress syndrome, chronicobstructive pulmonary disease, asthma and pulmonary fibrosis; to enhanceimmune tolerance and to combat assaults to the immune system such asthose associated with certain allergies or organ transplant rejection;treatment of dermatological diseases and conditions such as psoriasis,skin pigmentation depletion, acne, keloid formation and skin cancer;behavioral, central nervous system and neuronal disorders such asanxiety, depression, memory dysfunction, and neuropathic pain; and renalconditions or diseases such as the treatment of renal cachexia andnatriuresis.

In a further aspect, the present invention provides the use of atherapeutically effective amount of a melanocortin 4 receptor agonistcompound according formula (I) or formula (II) as defined hereinabove,or pharmaceutically acceptable salts thereof, for the manufacture of amedicament useful to modulate normalizing or homeostatic activities suchas ovarian weight, placental development, prolactin secretion, FSHsecretion, intrauterine fetal growth, parturition, spermatogenesis,gonadal development, thyroxin release, aldosterone synthesis andrelease, body temperature, blood pressure, heart rate, vascular tone,brain blood flow, blood glucose levels, sebum secretion, pheromonesecretion, motivation, learning and behavior, pain perception,neuroprotection, nerve growth, bone metabolism, bone formation and bonedevelopment.

It will be appreciated that therapeutic interventions addressing bothnormal physiological and pathophysiological processes which utilize themelanocortin receptors are also contemplated.

The compounds of formulas (I) or (II) are ligands for at least one ofthe melanocortin receptors (MC1-R, MC2-R, MC3-R, MC4-R and MC5-R) and aselection thereof were tested for their ability to act as a ligand inthe in vitro assay described below.

In some aspects, provided herein is also a method of evaluating asubject, e.g., for likely responsiveness to a MC4R agonist describedherein. In some embodiments, the method comprises acquiring informationabout the genotype of the subject.

In embodiments, the identification of the subject having a defect, e.g.,genetic defect, e.g., mutation, indicates that the subject is likely torespond (e.g., with an improvement in one or more symptoms) to a MC4Ragonist, e.g., a MC4R agonist described here. In embodiments, animprovement in a symptom can include an outcome described herein. Forexample, an improvement in a symptom can include a reduction of weight(e.g., body weight), a reduction in hunger level, no detectable decreasein energy expenditure (e.g., resting energy expenditure), an increase inenergy expenditure (e.g., resting energy expenditure), a reduction indaily/weekly/monthly food intake, or a reduction in waist circumference,e.g., relative to a control.

In embodiments, methods described herein further comprise providing areport that identifies the presence or absence of the genetic defect andin some cases an identifier for the subject. In embodiments, the reportprovides a recommendation on potential therapeutic options, likelyeffectiveness of a therapeutic option, and/orrecommendations/instructions for administration of the therapeuticoption (e.g., a MC4R agonist described herein).

MC4R Agonists

The invention provides MC4R agonists according to Formula I:

General Structure:

-   -   Aaa, Bbb=Selected from Cys, hCys, Pen; capable of establishing a        disulfide bridge or        -   Glu, Asp, Lys, Orn, Dpr, Dbu capable of establishing a            lactam bridge    -   Xxx=Asn, Gln, Ser, Thr    -   Yyy=Lys, Arg, D-Lys, D-Arg    -   A₁=H, Ac    -   A₂=OH, NH₂    -   Ac is acyl, e.g., acetyl.

In embodiments, an MC4R agonist has a structure according to Formula II:

where Xxx is Asn, Gln, Ser, or Thr,where A₁ is H or Ac,where A₂ is OH or NH₂, andwhere Yyy is Lys, Arg, D-Lys, or D-Arg.

In embodiments, the MC4R agonist is chosen from one or more of thefollowing compounds, (or pharmaceutically acceptable salt thereof):

001554C (SEQ ID NO: 1) Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ 001555C(SEQ ID NO: 2) Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ 001556C(SEQ ID NO: 3) Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ 001574C:(SEQ ID NO: 4) Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ 001576C:(SEQ ID NO: 5) Ac-Arg-(Glu-Gln-D-Phe-Arg-Trp-Apr)-NH₂ (SEQ ID NO: 6)Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 7)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 8)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 9)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 10)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 11)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 12)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 13)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 14)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 15)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 16)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 17)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 18)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 19)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 20)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 21)Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 22)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 23)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 24)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 25)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 26)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 27)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 28)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 29)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 30)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 31)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 32)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 33)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 34)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 35)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 36)Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 37)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 38)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 39)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 40)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 41)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 42)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 43)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 44)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 45)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 46)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 47)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 48)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 49)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 50)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 51)Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 52)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 53)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 54)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 55)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 56)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 57)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 58)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 59)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 60)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 61)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 62)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 63)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 64)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ or (SEQ ID NO: 65)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂.

EC-50 and Selectivity Ratios for exemplary compounds of the inventionare provided below:

001554C (SEQ ID NO: 1) Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ 001555C(SEQ ID NO: 2) Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ 001556C(SEQ ID NO: 3) Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ 001574C:(SEQ ID NO: 4) Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ 001576C:(SEQ ID NO: 5) Ac-Arg-(Glu-Gln-D-Phe-Arg-Trp-Apr)-NH₂

Administration of a compound or pharmaceutically acceptable salt thereofor a composition comprising a compound or pharmaceutical salt of acompound of the invention useful to practice the methods describedherein, can be continuous, hourly, four times daily, three time daily,twice daily, once daily, once every other day, twice weekly, onceweekly, once every two weeks, once a month, or once every two months, orlonger or some other intermittent dosing regimen.

Examples of administration of a compound or composition comprising acompound or pharmaceutical salt of a compound of the invention includeperipheral administration. Examples of peripheral administration includeoral, subcutaneous, intraperitoneal, intramuscular, intravenous, rectal,transdermal or intranasal forms of administration.

As used herein, peripheral administration can include all forms ofadministration of a compound or a composition comprising a compound ofthe instant invention which excludes intracranial administration.Examples of peripheral administration include, but are not limited to,oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous orsubcutaneous injection, extended release, slow release implant, depotand the like), nasal, vaginal, rectal, sublingual or topical routes ofadministration, including transdermal patch applications and the like.

The nomenclature used to define the peptides is that typically used inthe art wherein the amino group at the N-terminus appears to the leftand the carboxyl group at the C-terminus appears to the right. Where theamino acid has D and L isomeric forms, it is the L form of the aminoacid that is represented unless otherwise explicitly indicated.

The compounds of the invention useful for practicing the methodsdescribed herein may possess one or more chiral centers and so exist ina number of stereoisomeric forms. All stereoisomers and mixtures thereofare included in the scope of the present invention. Racemic compoundsmay either be separated using preparative HPLC and a column with achiral stationary phase or resolved to yield individual enantiomersutilizing methods known to those skilled in the art. In addition, chiralintermediate compounds may be resolved and used to prepare chiralcompounds of the invention.

The compounds described herein may exist in one or more tautomericforms. All tautomers and mixtures thereof are included in the scope ofthe present invention. For example, a claim to 2-hydroxypyridinyl wouldalso cover its tautomeric form, α-pyridonyl.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, all publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety.

Symbol Meaning Ac acyl group hCys Homocysteine Dpr 2,3-Diaminopropionicacid Dbu 2,4-Diaminobutyric acid Orn Ornithine

Unless otherwise indicated, with the exception of the N-terminal aminoacid, all abbreviations (e.g. Ala) of amino acids in this disclosurestand for the structure of —NH—C(R)(R′)—CO—, wherein R and R′ each is,independently, hydrogen or the side chain of an amino acid (e.g., R═CH₃and R′═H for Ala), or R and R′ may be joined to form a ring system.

Pharmaceutical Compositions/Administration

In accordance with any method or composition described herein, inembodiments, provided herein is a unit dosage of a MC4R agonistdescribed herein. In embodiments, the unit dosage contains 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, or 2 mg of the agonist. In embodiments, the unit dosage issuitable for injection, e.g., subcutaneous injection. In embodiments,the unit dosage is disposed in a delivery device suitable for injection,e.g., subcutaneous injection. In embodiments, the unit dosage isdisposed in a syringe suitable for injection, e.g., subcutaneousinjection, or a pen-type injector. Exemplary pen-type injectors aredescribed, e.g., in U.S. Pat. No. 8,512,297B2, U.S. Pat. No. 5,688,251A,U.S. Pat. No. 5,820,602A, US2014/0163526A1, and U.S. Pat. No.5,226,895A, incorporated herein by reference.

In embodiments, also provided herein is a pharmaceutical compositioncomprising a MC4R agonist described herein. In embodiments, thepharmaceutical composition includes a therapeutically effective amountof a MC4R agonist described herein. A therapeutically effective amountof the agonist can vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the agonistto elicit a desired response in the individual, e.g., amelioration of atleast one disorder parameter, e.g., a parameter of obesity orhyperphagia, or amelioration of at least one symptom of the disorder,e.g., obesity, hyperphagia, or Prader Willi Syndrome (PWS). Inembodiments, a therapeutically effective amount is also one in which anytoxic or a detrimental effect of the composition is outweighed by thetherapeutically beneficial effects.

In certain embodiments, the agonist may be prepared with a carrier thatwill protect it against rapid release, such as a controlled releaseformulation, including implants, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

In other embodiments, the agonist can be prepared using methodsdescribed in WO2014/144842, incorporated herein by reference. Inembodiments, the agonist is prepared in a formulation comprising ananionic excipient, e.g., PEG-carboxylic acid, fatty acid having 10 ormore carbon atoms, and/or anionic phospholipid. In embodiments, theanionic phospholipid is described in WO2014/144842 (e.g., at pages 7-9).In some embodiments, the anionic phospholipid is1,2-distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE), optionallyconjugated to polyethylene glycol (PEG), the structure of which is:

with the value of “n” varying with molecular weight. In embodiments, thefatty acid is described in WO2014/144842 (e.g., at page 9). Inembodiments, the PEG-carboxylic acid is described in WO2014/144842(e.g., at pages 9-11). In embodiments, the molar ratio of the agonist tothe anionic excipient ranges from about 1:1 to about 1:10.

In embodiments, the agonist forms an ionic complex with the othercomponents of the formulation, and e.g., provides a desirablepharmacokinetic profile for the agonist (e.g., extend duration of drugaction and/or minimize adverse effects). In embodiments, the formulationis a sustained release formulation. In embodiments, the formulationprovides reduced fluctuations in concentration of the agonist afteradministration.

A MC4R agonist described herein, can be administered to a subject, e.g.,human subject, by various methods. In embodiments, the route ofadministration is one of: intravenous injection or infusion,subcutaneous injection, or intramuscular injection. In embodiments, theroute of administration is subcutaneous injection.

In embodiments, pharmaceutical compositions, e.g., comprising a MC4Ragonist described herein, can be administered with medical devices. Forexample, compositions comprising the agonist can be administered with aneedleless hypodermic injection device, such as the devices disclosed inU.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880,4,790,824, or 4,596,556. Examples of implants and modules include: U.S.Pat. No. 4,487,603, which discloses an implantable micro-infusion pumpfor dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicamentsthrough the skin; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Other such implants, delivery systems, and modulescan also be used.

In embodiments, continuous administration can be indicated, e.g., viasubcutaneous pump. In embodiments, the agonist is administered via asyringe (e.g., prefilled syringe), an implantable device, a needlelesshypodermic injection device, an infusion pump (e.g., implantableinfusion pump), or an osmotic delivery system.

In embodiments, the agonist is administered at a unit dosage, e.g.,comprising 0.1-10 mg, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg of the agonist, e.g.,subcutaneously.

In embodiments, the agonist is administered in a bolus at a dose ofbetween 0.1-10 mg, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg of the agonist, e.g.,subcutaneously.

In embodiments, the agonist is administered continuously, e.g., via apump, e.g., subcutaneous pump.

In embodiments, the agonist, e.g., a unit dosage of the agonist, isdisposed within a delivery device, e.g., a syringe (e.g., prefilledsyringe), an implantable device, a needleless hypodermic injectiondevice, an infusion pump (e.g., implantable infusion pump), or anosmotic delivery system.

In embodiments, a daily dosage of the agonist is administered, e.g.,subcutaneously, to a subject. In embodiments, the daily dosage of theagonist is about 0.1 mg to about 10 mg, e.g., 0.1-0.2, 0.2-0.4, 0.4-0.6,0.6-0.8, 0.8-1, 1-1.2, 1.2-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4,4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9,9-9.5, 9.5-10 mg, e.g., administered subcutaneously.

In embodiments, the agonist, is administered, e.g., via one or multipleadministrations, over a period of at least 3 weeks, e.g., at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40weeks or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 10, 11, or 12months or more, or at least 1, 2, 3, 4 years or more. In embodiments,where multiple administrations are provided of the agonist, the timeinterval in between any two of the administrations is at least 6 hours,e.g., 6 h, 12 h, 24 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more. In embodiments, theinterval in between any two of the administrations is 1 day.

Kits

A MC4R agonist described herein can be provided in a kit. The kitincludes a MC4R agonist described herein and, optionally, a container, apharmaceutically acceptable carrier and/or informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the agonist for the methods described herein.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the agonist, physical properties of the agonist,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods for administering the agonist, e.g., by a route ofadministration described herein and/or at a dose and/or dosing scheduledescribed herein.

In one embodiment, the informational material can include instructionsto administer an agonist described herein in a suitable manner toperform the methods described herein, e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer an agonistto a suitable subject, e.g., a human, e.g., an obese human, e.g.,severely obese human.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about an agonistdescribed herein and/or its use in the methods described herein. Theinformational material can also be provided in any combination offormats.

In addition to an agonist, the composition of the kit can include otheringredients, such as a surfactant, a lyoprotectant or stabilizer, anantioxidant, an antibacterial agent, a bulking agent, a chelating agent,an inert gas, a tonicity agent and/or a viscosity agent, a solvent orbuffer, a stabilizer, a preservative, a pharmaceutically acceptablecarrier and/or a second agent for treating a condition or disorderdescribed herein. Alternatively, the other ingredients can be includedin the kit, but in different compositions or containers than an agonistdescribed herein.

In some embodiments, a component of the kit is stored in a sealed vial,e.g., with a rubber or silicone closure (e.g., a polybutadiene orpolyisoprene closure). In some embodiments, a component of the kit isstored under inert conditions (e.g., under Nitrogen or another inert gassuch as Argon). In some embodiments, a component of the kit is storedunder anhydrous conditions (e.g., with a desiccant). In someembodiments, a component of the kit is stored in a light blockingcontainer such as an amber vial.

An agonist described herein can be provided in any form, e.g., liquid,frozen, dried or lyophilized form. It is preferred that a compositionincluding the agonist described herein be substantially pure and/orsterile. When an agonist described herein is provided in a liquidsolution, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being preferred. In one embodiment, the agonistis supplied with a diluents or instructions for dilution. The diluentcan include for example, a salt or saline solution, e.g., a sodiumchloride solution having a pH between 6 and 9, lactated Ringer'sinjection solution, D5W, or PLASMA-LYTE A Injection pH 7.4® (Baxter,Deerfield, Ill.).

The kit can include one or more containers for the compositioncontaining an agonist described herein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, IV admixture bag, IV infusion set,piggyback set or syringe (e.g., prefilled syringe), and theinformational material can be contained in a plastic sleeve or packet.In other embodiments, the separate elements of the kit are containedwithin a single, undivided container. For example, the composition iscontained in a bottle, vial or syringe that has attached thereto theinformational material in the form of a label. In embodiments, thecomposition is contained in an injector device, e.g., a pen-typeinjector. The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: Synthesis of Polypeptides of Formula (I and II)

The polypeptides of Formula (I and II) were prepared by conventionalsolid phase peptide synthesis. The peptide chain was elongated in astep-wise manner starting with its C-terminal end amino acid derivativecoupled on to a suitably selected solid support resin known to besuitable for peptide synthesis. For the synthesis of peptide with aC-terminal amide function, Rink amide MBHA resin was employed as solidsupport. For the synthesis of peptides with the C-terminal free carboxylfunction, resins such as 2-chlorotrityl chloride resin, Wang, orMerrifield resin may be utilized that form an ester bond with theFmoc-amino acid. Most of these ester linked Fmoc-amino acid-resin typesare commercially available from various sources and generally used whenfeasible.

Synthesis of Disulfide-Cyclized Peptides

The linear derivative of a disulfide cyclic peptides amide was assembledusing Fmoc chemistry on a solid-phase peptide synthesizer. The Fmoc-Rinkamide resin was placed in a reaction vessel and swollen with NMP. It wasthen treated with 20% piperidine in NMP for 15 minutes, followed by 3washes of NMP. The resin was tested for positive Kaiser's test (Kaiser,E., Colescot, R. L., Bossinge, C. D. & Cook, P. I. Anal. Biochem., 1990,34: 595-598). It was resuspended in NMP and mixed with the requiredfirst C-terminal Fmoc-amino acid derivative and HOBt. The couplingreaction was started by the addition of HBTU reagent and DIPEA. Aftermixing for 2-3 hours, the completion of coupling was confirmed by anegative Kaiser's test on a small aliquot of the resin withdrawn fromthe reaction mixture. The resin was then washed three times with NMP.Thereafter, the Fmoc group was removed as described earlier and thewhole cycle repeated with the second C-terminal Fmoc-amino acidderivative as described. The same cycle of reactions was repeatedsequentially with each of the incoming amino acid. In the case ofpeptides with an N-terminal acetyl group, the Fmoc deprotected peptideresin was treated for 10 minutes with acetic anhydride and pyridine. Theresin after testing negative for Kaiser's test was washed with NMP,dichlromethane and dried in vacuo. The Fmoc-amino acid derivatives wereused for the synthesis of these peptides. The trifunctional amino acidderivatives used were the following: Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-hCys(Trt)-OH,Fmoc-Pen(Trt)-OH, Fmoc-Glu(OBut)-OH, and Fmoc-D-Phe-OH.

To cleave the peptide off the resin as well as deprotect the side chainfunctions, the peptide resin was taken in 2% TIS/5% water/5% (w/v)DTT/88% TFA. The solution was allowed to mix for 3.5 hours and thenfiltered. The filtrate was mixed with cold anhydrous ethyl ether. Theprecipitate was collected by centrifugation. The solvent was decantedand the peptide pellet was re-suspended in fresh ether. The ether workupwas repeated two more times. The peptide was dried in vacuo. The crudelinear peptide product was diluted to a concentration of 2 mg/mL in 5%acetic acid and 0.5M iodine/methanol was added dropwise with vigorousstirring until a persistent pale yellow color of the solution wasachieved. The solution was stirred for additional 10 minutes. Excessiodine was then quenched by adding 1M sodium thiosulfate under mixinguntil the mixture was rendered colorless. The cyclized peptide solutionwas lyophilized and the crude powder purified by preparative HPLC usinga reversed-phase C-18 column. The purified product fractions were pooledand lyophilized.

The peptides were analyzed by mass spectrometry using electrosprayionization technique and identified to correct mass.

Synthesis of Lactam-Cyclized Peptides

The cyclic lactam peptides were also synthesized by standard solid phasepeptide synthesis methods. For peptides with a C-terminus Dpr, anFmoc-Dpr(Mtt)-BHA and for peptides with a C-terminal Abu, anFmoc-Abu(Mtt)-BHA resin was transferred to a solid phase peptidesynthesizer reactor. The Fmoc group, was removed as described above andthe next Fmoc-protected amino acid, such as for exampleFmoc-Trp(Boc)-OH, was coupled to the resin through standard couplingprocedures. The Fmoc protective group was removed and the remainingamino acids added individually in the correct sequence, by repeatingcoupling and deprotection procedures until the amino acid sequence wascompleted. For glutamic acid or aspartic acid coupling Fmoc-Glu(OPip) orFmoc-Asp(OPip) was employed. The fully assembled peptide was thenacetylated at the N-terminus as per method described earlier for thedisulfide series of peptides. The orthogonally protected side chainswere then removed. For example, a peptide resin with either anorthogonally protected side chain of Glu as 2-phenylisoproply (OPip)ester or Dpr as 4-methyltrityl (Mtt), were cleaved by treatment with 1%TFA in dicholoromethane. The deprotected peptide resin was suspended inNMP, and treated with HBTU/DIPEA. After cyclization (a negative Kaiser'stest), the peptide-resin was washed with DCM and dried. The cyclicpeptide was cleaved from the resin along with any remaining protectivegroups using trifluoroacetic acid (TFA) in the presence of water and1,2-ethanedithiol (EDT). The product was collected by precipitation uponthe addition of cold anhydrous ether and collected by centrifugation.Final purification was by reversed phase HPLC using a reversed phaseC-18 column. The purified peptide collected by lyophilization andanalyzed for its mass by mass spectrometry using electron spraymethodology.

One skilled in the art can modify the synthesized polypeptides ofFormula (I and II) described herein.

Radioligand Binding Assays:

Receptor binding assays for determining the binding constant (K_(d)) orinhibition concentration (IC₅₀) for displacing a radio-labeled ligandfrom the receptor of a cyclic peptide of Formula (I or II) may beconducted by any means known in the art.

As an example, the cell membrane preparations for a binding assay areprepared from CHO-K1 cells transfected to stably express hMC receptorsubtypes 1, 3, 4 or 5. Competitive inhibition of[¹²⁵I](Tyr²)-(Nle⁴-D-Phe⁷)-alpha-MSH ([¹²⁵I]-NDP-α-MSH binding iscarried out in polypropylene 96 well plates. Briefly, the cell membranes(1-10m protein/well), prepared as described above, is incubated in 50 mMTris-HCl at pH 7.4 containing 0.2% BSA, 5 mM MgCl₂, 1 mM CaCl₂ and 0.1mg/mL bacitracin, with increasing concentrations of the test compoundand 0.1-0.3 nM [¹²⁵I ]-NDP-α-MSH for approximately 120 minutes at 37° C.Bound [¹²⁵I]-NDP-α-MSH ligand is separated from free [¹²⁵I]-NDP-α-MSH byfiltration through GF/C glass fiber filter plates (Unifilter®, Meriden,Conn., USA) presoaked with 0.1% (w/v) polyethylenimine (PEI). Filtersare washed three times with 50 mM Tris-HCl at pH 7.4 at a temperature ofapproximately 0-4° C. and then assayed for radioactivity. The bindingdata are analyzed by computer-assisted non-linear regression analysis.

Cyclic AMP Stimulation Assay

Functional assays to determine agonist or antagonist status of a cyclicpeptide of polypeptide of Formula (I and II) are performed using methodsknown in the art.

Electrochemiluminescence (ECL) Assay

Stimulation of intracellular cyclic AMP (cAMP) levels by the peptides isdetermined in a dose dependent manner by an electrochemiluminescence(ECL) assay (Meso Scale Discovery, Gaithersburg, Md., USA; referred tohereinafter as “MSD”). Briefly, the CHO-K1 cells stably expressing thehMC receptor subtypes are suspended in RMPI 1640® assay buffer (RMPI1640 buffer contains 0.5 mM IBMX, and 0.2% protein cocktail (MSD blockerA)). About 7,000 cells/well of the transgenic CHO-K1 cells stablyexpressing hMC receptor subtypes 1, 3, 4 or 5 are dispensed in 384-wellMulti-Array plates (MSD) containing integrated carbon electrodes andcoated with anti-cAMP antibody. Increasing concentrations of the testcompounds are added and the cells are incubated for approximately 40minutes at 37° C. A cell lysis buffer (HEPES-buffered saline solutionwith MgCl₂ and Triton X-100® at pH 7.3) containing 0.2% protein cocktailand 2.5 nM TAG™ ruthenium-labeled cAMP (MSD) is added and the cells areincubated for approximately 90 minutes at room temperature. At the endof the second incubation period, the read buffer (Tris-buffered solutioncontaining an ECL co-reactant and Triton X-100 at pH 7.8) is added andthe cAMP levels in the cell lysates are immediately determined by ECLdetection with a Sector Imager 6000 Reader® (MSD). Data are analyzedusing a computer-assisted non-linear regression analysis (XL fit; IDBS)and reported as either an EC50 value. The EC50 represents theconcentration of an agonist compound needed to obtain 50% of the maximumreaction response, e.g., 50% of the maximum level of cAMP as determinedusing the assay described above.

cAMP Measurement Assay

Human MC4-R transfected cells are grown to confluence in 96 well plates(plating approximately 250,000 cells per well). The cells are treated intriplicate sets with 0.2 mM isobutylmethylxanthine (IBMX) and gradedconcentrations of the peptide or alternatively the peptide in thepresence of 20 nM NDP-MSH. Cells similarly treated but with only 20 nMNDP-MSH serve as positive controls in a volume of 200 μL. A buffer blankserving as a negative control is also included. After incubation of onehour at 37° C., the cells are lysed by the addition of 50 μL of a celllysis buffer. Total cAMP accumulated in 250 μL of this incubation mediumis quantitated using a commercially available low pH cAMP assay kit(Amersham BioSciences) as per procedure specified by the kit supplier. Apeptide showing cAMP accumulation in the range same or higher than thealpha-MSH as positive control is considered to be an agonist. The datafor agonist is plotted and curve fitted to determine the EC50 value. Apeptide showing accumulation in the same range as the negative control(buffer blank in the absence of alpha-MSH) is ineffective at the testconcentration. A peptide showing attenuated accumulation is consideredto be an antagonist if there is inhibition in cAMP when alpha-MSH isalso present in the assay. Similar assay can be performed with hMC-1R,hMC-3R, and hMC-5R cells.

cAMP Accumulation Measurement Via a β-Galactosidase (β-Gal) ReporterSystem

A chemiluminescence readout system that uses an enzyme fragmentcomplementation (EFC) system with β-galactosidase (β-Gal) as thefunctional reporter system was used. This assay system for variousmelanocortin receptor systems is commercially available (cAMP HunterGPCR assay system, Discoverx Corp, Fremont, Calif.). This assay utilizesthe β-Gal enzyme that is split into two complementary portions; EA forEnzyme Acceptor and ED for Enzyme Donor. In the assay, the ED portionfused to cAMP is made to compete with cAMP generated by cells forbinding to a cAMP-specific antibody. The EA is then added to form activeβ-Gal with any unbound ED-cAMP. This active enzyme then converts achemiluminescent substrate to generate an output signal that is recoredon a standard microplate reader.

Briefly, 10000 cells per well are plated overnight and each well (cellsincubated with 10 μl assay buffer) is then incubated with 4× serialconcentration of the test compound in the cell assay buffer (5 μL) andcAMP antibody reagent (5 μL) for 30 min at 37° C. The cell lysis buffer(20 μL) containing ED-cAMP coupled enzyme fragment and the reportersubstrate (Emerald II-Galacton Star, 5:1) is then added and incubatedfor 60 min at room temperature. Next, 20 μL of EA β-Gal fragment reagentis added. After further incubation for 120 min at room temperature, thechemiluminescence is measured by a plate reader (Envision).

The EC-50 data for exemplary polypeptides of structural formulas I andII are shown in below.

Example: EC50 values and selectivity.

Selectivity Ratios Compound EC-50 (nM) MC4R/ MC4R/ MC4R/ ID MC-1 R MC-3RMC-4R MC-5R MC1R MC3R MC5R 001554C 235.06 >3000 1.60 735.47146.75 >1872.97 459.17 SEQ ID NO: 1 001555C 47.49 0.52 0.19 124.33244.35 2.68 639.81 SEQ ID NO: 2 001556C 228.52 >3000 1.86 1701.86123.06 >1615.55 916.48 SEQ ID NO: 3 001574C 516.38 >3000 1.11 2047.30465.77 >2705.95 1846.64 SEQ ID NO: 4 001576C 23.59 1.16 0.66 67.26 35.571.75 101.41 SEQ ID NO: 5 RM-493 1.80 1.06 0.94 1684.02 1.92 1.13 1799.06Melanotan 0.28 1.33 3.08 80.48 0.09 0.43 26.11 II

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

1. A compound of formula I:

where Xxx is Asn, Gln, Ser, or Thr, where A₁ is H or Ac, where A₂ is OHor NH₂, and where Yyy is Lys, Arg, D-Lys, or D-Arg, or apharmaceutically acceptable salt, single stereoisomer, mixture ofstereoisomers, ester, tautomer or prodrug thereof.
 2. The compound ofclaim 1 wherein the compound is a compound of Formula II.

where Xxx is Asn, Gln, Ser, or Thr, where A₁ is H or Ac, where A₂ is OHor NH₂, and where Yyy is Lys, Arg, D-Lys, or D-Arg.
 3. The compound offormula 1 wherein the compound comprises the sequence of any of SEQ IDNOs:1-64.
 4. A pharmaceutical composition comprising a compound of anyof claims 1-3 and a pharmaceutically acceptable carrier.
 5. A unitdosage any of the compounds of claims 1-3, wherein the unit dosagecontains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 100 mg ofthe compound.
 6. The unit dosage of claim 5, which contains 0.5 mg, 1.0mg, or 1.5 mg of the compound.
 7. The unit dosage of any of claims 5-6,suitable for injection, e.g., subcutaneous injection.
 8. The unit dosageof any of claims 5-7, disposed in a delivery device suitable forinjection, e.g., subcutaneous injection.
 9. A method of treating acondition or disorder in a subject in need thereof, comprisingadministering a therapeutically effective amount of an MC4R agonist ofany of claims 1-3.
 10. The method of claim 9, comprising eliciting anagonist effect from a melanocortin receptor in the subject.
 11. Themethod of claim 9, wherein administering the MC4R agonist elicits anagonist effect from the melanocortin receptor in the subject.
 12. Themethod of claim 9, wherein the disorder is a metabolic disease ormedical condition accompanied by weight gain such as obesity, feedingdisorders or Prader-Willi Syndrome.
 13. The method of claim 9, whereinthe MC4R agonist is administered in an amount sufficient to decreasefood intake, decrease body weight, or both, in the subject.
 14. Themethod of claim 9, wherein the MC4R agonist is administered in an amountsufficient to decrease appetite in the subject without compromising bodyweight.
 15. The method of claim 9, wherein the MC4R agonist isadministered in amount sufficient to ameliorate or improve a clinicalsymptom of obesity, type-II diabetes, a pre-diabetic condition, a bloodlevel of hemoglobin A1C (Hb1Ac) above 6%, hyperinsulimenia,hyperlipidemia, insulin insensitivity, or glucose intolerance; aspectsof metabolic syndrome, delaying, inhibiting or preventing theprogression of obesity and/or obesity related indications; or partiallyor totally delaying, inhibiting or preventing the onset or developmentof obesity or a obesity related indication.
 16. The method of claim 9,wherein the condition or disorder is obesity or a disorder associatedwith obesity, e.g., Prader-Willi Syndrome, decreasing food intake in asubject in need thereof, or decreasing body weight.
 17. The method ofany of the preceding claims, comprising administering the agonist in aunit dosage suitable for injection, e.g., subcutaneous injection, to thesubject.
 18. The method of claim 6, wherein the unit dosage is disposedwithin a delivery device, e.g., a syringe (e.g., prefilled syringe), animplantable device, a needleless hypodermic injection device, aninfusion pump (e.g., implantable infusion pump), or an osmotic deliverysystem.
 19. The method of any of the preceding claims, wherein theagonist is administered subcutaneously, e.g., by subcutaneous injection.20. The method of any of the preceding claims, wherein the agonist isadministered daily over a period of at least 3 weeks, e.g., at least 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 weeks or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12months or more, or at least 1, 2, 3, 4 years or more.
 21. The method ofany of the preceding claims wherein administration of the compounddecreases body weight in the subject.
 22. The method of any of thepreceding claims, wherein the subject is obese, e.g., severely obese.23. The method of any of the preceding claims, wherein the subject hasearly onset severe obesity.
 24. The method of any of the precedingclaims, wherein the subject is hyperphagic.
 25. The method of any of thepreceding claims, wherein the subject has a body mass index (BMI)greater than 25 kg/m² (e.g., ≥25, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 kg/m² or greater) priorto administration of the agonist, e.g., at the time the agonist isprescribed, or at the time of the first administration.
 26. The methodof any of the preceding claims, wherein the subject has failed one ormore previous therapies, e.g., exercise, diet, bariatric surgery orvertical banded gastroplasty, or behavioral therapies, prior toadministration of the agonist, e.g., at the time the agonist isprescribed, or at the time of the first administration.
 27. The methodof any of the preceding claims, wherein administration of the agonistresults in a reduction of food intake by the subject compared to acontrol (e.g., the food intake of the subject prior to treatment), e.g.,wherein the food intake is daily food intake over a period of 24 hours,or one week.
 28. The method of any of the preceding claims, wherein theagonist comprises the sequence 001554C (SEQ ID NO: 1)Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ 001555C (SEQ ID NO: 2)Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ 001556C (SEQ ID NO: 3)Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ 001574C: (SEQ ID NO: 4)Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ 001576C: (SEQ ID NO: 5)Ac-Arg-(Glu-Gln-D-Phe-Arg-Trp-Apr)-NH₂ (SEQ ID NO: 6)Ac-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 7)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 8)H-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 9)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 10)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 11)Ac-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 12)H-D-Arg-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 13)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 14)Ac-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 15)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 16)H-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 17)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 18)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 19)Ac-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 20)H-D-Lys-(hCys-Asn-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 21)Ac-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 22)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 23)H-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 24)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 25)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 26)Ac-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 27)H-D-Arg-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 28)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 29)Ac-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 30)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 31)H-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 32)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 33)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 34)Ac-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 35)H-D-Lys-(hCys-Gln-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 36)Ac-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 37)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 38)H-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 39)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 40)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 41)Ac-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 42)H-D-Arg-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 43)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 44)Ac-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 45)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 46)H-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 47)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 48)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 49)Ac-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 50)H-D-Lys-(hCys-Ser-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 51)Ac-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 52)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 53)H-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 54)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 55)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 56)Ac-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 57)H-D-Arg-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 58)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 59)Ac-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 60)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 61)H-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-OH (SEQ ID NO: 62)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 63)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ (SEQ ID NO: 64)Ac-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂ or (SEQ ID NO: 65)H-D-Lys-(hCys-Thr-D-Phe-Arg-Trp-Pen)-NH₂.